HIV/AIDS continues to be a major threat to human health. The use of combinations of small molecule drugs in highly active anti-retroviral therapy (HAART) to stop or thwart HIV propagation ahs had a major impact on delaying the progression from HIV-1 infection to the development of AIDS. Despite this progress, there are problems associated with a lifelong use of anti-viral drug therapy. These include toxicity, the emergence of virus resistant to multiple drugs, and the cost of a daily medication. The proposed studies take advantage of recent advances in gene therapy for the treatment of HIV-1 infection. Gene therapy of human blood progenitor cells can be considered as a potential replacement or supplement to the current anti-HIV-1 therapies. An outcome of the previous funding period was the development of a three pronged genetic approach for inhibiting viral entry and replication. This triple small RNA gene therapy has been tested for feasibility and safety in a first in human blood stem cell gene therapy trial in AIDS/lymphoma patients. Based upon the results of this four patient trial we believe it is imperative to progress to new and different small RNA anti-viral combinations. The proposed studies take advantage of a set of novel inhibitors of HIV-1 infection which use different mechanisms of action. We hypothesize that such combinations will prevent the emergence of viral resistant mutants and at the same time be non-toxic. Specific Aim 1 will select for in vitro evolved RNA aptamers that selectively bind HIV-1 integrase and block integrase function. The aptamers will be expressed in hematopoietic cells as part of a combinatorial approach designed to block viral infection. Specific Aim 2 will combine nucleolar localizing anti-HIV RNAs, microRNA mimics, tRNA-anti CCR5 shRNA, U5 tRNA shRNA fusions and the anti-integrase aptamers in a lentiviral vector for transduction of hematopoietic progenitor cells. The results from this research will provide novel combinations of anti-HIV RNAs for functional testing in vivo. Specific Aim 3 addresses the in vivo analyses of combinatorial constructs in the humanized Rag2-/- 3c-/- (RAG-hu) mouse model. This humanized mouse model represents a novel and facile system for testing the efficacy and safety of anti-HIV gene therapy strategies since gene modified human hematopoietic stem and progenitor cells can engraft and differentiate into hematopoietic lineages that are infectible by HIV-1 in vivo. Overall, this program is designed to be a path towards bringing new small RNA based gene therapy combinations to the clinic for use in future human gene therapy applications.